Project Summary/Abstract Discovery of an antithrombotic therapy that does not cause bleeding would be a transformative advance in the management of millions of patients with conditions such as myocardial infarction, thromboembolic disease, and stroke who require anticoagulation but are put at risk of major hemorrhage with currently available medications. One highly promising target is coagulation factor XII (FXII/FXIIa), based on the recent finding that mice lacking FXII are strikingly protected from thrombosis without increased bleeding. Because it has long been known that severe congenital FXII deficiency in humans does not cause bleeding, these results make FXII a potentially groundbreaking antithrombotic target that could ?decouple? efficacy from bleeding complications. As proof of concept, we have shown that X210-C01, a novel therapeutic antibody against murine FXIIa, prevents arterial thrombus formation in mice while preserving hemostasis. However, the mechanism by which FXII is recruited, activated, and propagated during arterial thrombosis but not hemostasis remains poorly understood. Our central hypothesis is that platelets bind and activate FXII in arterial thrombi but not in hemostatic plugs, after which FXIIa is amplified by positive feedback via a plasma cofactor. To define the molecular mechanisms underlying FXII function in vivo, we propose the following aims: 1) Test whether plasma kallikrein (PK) is an essential cofactor for FXIIa in thrombus formation. We will use a novel anti-PK antibody (M202-H03) to evaluate the effect of dual PK and FXIIa blockade on thrombus formation in vivo. We will also study the mechanism by which PK interacts with and activates FXII. 2) Define mechanisms of FXII activation during thrombus formation in vivo. We will test whether phosphatidylserine and platelet GPIb? are necessary and sufficient to activate FXII at the platelet surface. We will then use intravital confocal microscopy to localize FXII during thrombus formation. 3) Using in vivo models, test whether FXII inhibition impairs hemostasis and determine if platelets in hemostatic plugs are phenotypically distinct from those in arterial thrombi. The applicant, Dr. Pavan Bendapudi, is well qualified to execute the proposed experiments. He is committed to pursuing a scientific career in hemostasis and thrombosis and has proposed a comprehensive five-year plan to meet his goal of becoming an independent physician-scientist. Dr. Bendapudi will be working under the primary mentorship of Dr. Robert Flaumenhaft with Dr. Bruce Furie serving as co-mentor. He has enlisted a research advisory committee of internationally-recognized experts in hematology to support him. The Division of Hemostasis and Thrombosis at Beth Israel Deaconess Medical Center is an ideal environment for completion of his scientific and career development objectives given its outstanding research community and tradition of scientific discovery and trainee mentorship in this field.